CN114317703A

CN114317703A - Nucleic acid sequence determination method, system, storage medium, and computer program product

Info

Publication number: CN114317703A
Application number: CN202011052677.1A
Authority: CN
Inventors: 万雪峰; 吴平; 姜泽飞
Original assignee: Genemind Biosciences Co Ltd
Current assignee: Genemind Biosciences Co Ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-04-12

Abstract

Disclosed are a method of determining a nucleic acid sequence, a nucleic acid sequence determination system, a computer-readable storage medium, and a computer program product, the method of determining a nucleic acid sequence comprising: rotating a rotary valve provided with a first flow passage and a second flow passage to a first valve position to communicate a first storage and a reaction device; under the condition that the rotary valve is in the first valve position, enabling the first reaction liquid to enter the reaction device through the first flow channel to perform a first reaction; rotating the rotary valve to a second valve position to communicate the second reservoir with the reaction device; with the rotary valve in the second valve position, a second reaction solution is caused to enter the reaction device through the second flow path to perform a second reaction to effect sequencing of the nucleic acid molecule. Therefore, the rotary valve is positioned at different positions, so that the flow directions of different liquids can be independently controlled and operated, and the sequence determination of nucleic acid molecules is easier to realize.

Description

Nucleic acid sequence determination method, system, storage medium, and computer program product

Technical Field

The present application relates to the field of nucleic acid sequence determination technology, and more particularly, to a method for determining a nucleic acid sequence, a nucleic acid sequence determination system, a storage medium, and a computer program product.

Background

In the related art, nucleic acid detection of a nucleic acid sample disposed on a solid substrate, for example, nucleic acid sequencing (sequencing) based on chip detection, generally includes sample processing before loading and loading, where the sample processing before loading is generally referred to as processing a nucleic acid sample to be detected so as to meet the loading requirement, for example, attaching the nucleic acid sample to the surface of the solid substrate, and the loading refers to entering the processed nucleic acid sample to be detected into a sequencer for sequencing.

Current commercial automated sequencing platforms typically include two or more separate or functionally linked devices to accomplish the above-described sample processing and sequencing. For a sequencing platform comprising two mating devices, typically one of the devices is used for processing a nucleic acid sample to be tested, including attaching it to a reaction device such as a chip, the device is often referred to as a hybridization apparatus, a sample introduction apparatus, a library construction apparatus, a sample processing apparatus, or a sample preparation apparatus; the other device detects the chip connected with the nucleic acid sample to be detected and output by the last device, thereby realizing the determination of the nucleic acid sequence, and the device is the most main hardware of the sequencing platform, namely a sequencing instrument generally.

Current commercial sequencing platforms, these stand-alone and functionally linked devices can be sold or purchased together or separately/in part; purchasing multiple associated devices is generally expensive, and purchasing one or a portion of the devices, such as purchasing only one sequencer, typically requires more user operations or involvement in manual operations or selection of a commercial sample processing service.

With the development of sequencing technology, automation and other related technologies and the expectation of the market on simple operation and the like, a sequencer integrating at least a part of pre-computer sample processing and sequencing functions is a development direction (hereinafter, the sequencer with the integrated function is referred to as an integrated sequencer or an integrated sequencing platform for short), and particularly, compared with the sequencing platform before integration, the integrated sequencer is not weakened in performance, equivalent or smaller in volume and simpler and more convenient to operate.

Few currently mature integrated sequencer commodities with integrated functions are limited by at least the following factors: sample processing and sequencing before loading generally comprises a plurality of steps and a plurality of reactions, and relates to switching of a plurality of reagents, a plurality of serial or parallel liquid inlet and outlet sequence control and microfluidic control.

Therefore, how to design the corresponding system including a control system to realize multiple reactions, including switching of multiple reagents, and multiple serial or parallel liquid inlet and outlet sequence control, so that the corresponding actions and reactions can be orderly and automatically performed and the desired results can be obtained, is a considerable problem.

Disclosure of Invention

Embodiments of the present application provide a method of determining a nucleic acid sequence, a nucleic acid sequence determination system, a computer-readable storage medium, and a computer program product to address, at least to some extent, at least some of the problems discussed above.

The method for determining a nucleic acid sequence of the present embodiment comprises: rotating a rotary valve provided with a first flow channel and a second flow channel to a first valve position so as to communicate a first storage and a reaction device, wherein the first storage carries a first reaction liquid, and the first reaction liquid contains nucleic acid molecules; with the rotary valve in the first valve position, allowing a first reaction solution to enter the reaction device through the first flow channel to perform a first reaction, the first reaction comprising attaching at least a portion of the nucleic acid molecule to the reaction device; rotating the rotary valve to a second valve position to communicate a second storage and the reaction device, wherein the second storage carries a second reaction solution, and the second reaction solution contains components required for nucleic acid sequencing; and allowing the second reaction solution to enter the reaction device through the second flow channel with the rotary valve in the second valve position to perform a second reaction, the second reaction including allowing the nucleic acid molecule in the reaction device after the first reaction to interact with the second reaction solution to perform a polymerization reaction and detecting a signal from the reaction, the polymerization reaction occurring and detecting the signal from the reaction, to thereby effect sequencing of the nucleic acid molecule.

The method realizes the progress of various reactions by designing and controlling a liquid path system, which comprises a rotary valve comprising a plurality of independent flow channels and controlling the rotary valve to be at a first valve position or a second valve position so as to utilize the appointed flow channel to communicate a solution storage and a reaction device; the method integrates and simplifies the liquid path system of various reactions by configuring a specific rotary valve, so that the rotary valve or the liquid path system comprising the rotary valve can be simply operated to realize various reactions, and the method is particularly suitable for an automatic biomolecule detection system related to various reactions, such as a nucleic acid sequencing system.

The method can realize the nucleic acid Sequencing related to the solid phase carrier by enabling the rotary valve to be in a specified valve position and communicated with a specified flow channel to carry out various reactions, and comprises the steps of connecting or fixing the nucleic acid molecules on the surface of the solid phase carrier and carrying out Sequencing on the nucleic acid molecules on the surface of the solid phase carrier by Sequencing By Synthesis (SBS) or Sequencing By Ligation (SBL). The hardware configuration required for automatically realizing the method is simple and easy to realize, and is beneficial to batch preparation and industrialization.

A nucleic acid sequence measuring system according to an embodiment of the present application includes a fluid path system, a detection module, and a controller connected to each other, the fluid path system including a fluid network and a pump module connected to the fluid network, the fluid network including a first reservoir, a second reservoir, a rotary valve, and a reaction device, the rotary valve being provided with a first flow path and a second flow path, the first flow path communicating with the first reservoir and the reaction device, the second flow path communicating with the second reservoir and the reaction device, the detection module being configured to detect a signal from the reaction device during a prescribed reaction, and the controller being configured to control the fluid path system and the detection module to perform the steps of the nucleic acid sequence measuring method according to any of the above embodiments.

A computer-readable storage medium of an embodiment of the present application stores a program for execution by a computer, the program being executed to perform a method for performing nucleic acid sequence determination in any of the above embodiments.

The present application provides a system configured to perform the nucleic acid sequence determination method of any of the above embodiments.

A computer program product comprising instructions for carrying out the method of any one of the preceding embodiments when the program is executed by a computer.

A nucleic acid sequence determination system of an embodiment of the present application includes the computer program product of any of the above embodiments.

A nucleic acid sequence determination system according to an embodiment of the present application includes: the first control module is used for rotating the rotary valve provided with the first flow channel and the second flow channel to a first valve position so as to communicate the first storage and the reaction device, the first storage bears a first reaction liquid, and the first reaction liquid contains nucleic acid molecules; a second control module for allowing the first reaction solution to enter the reaction device through the first flow channel to perform a first reaction under the condition that the rotary valve is in the first valve position, wherein the first reaction comprises connecting at least a part of the nucleic acid molecules to the reaction device; the third control module is used for rotating the rotary valve to a second valve position so as to communicate the second memory and the reaction device, the second memory bears a second reaction liquid, and the second reaction liquid contains components required for nucleic acid sequencing; and the fourth control module is used for enabling the second reaction liquid to enter the reaction device through the second flow channel to carry out a second reaction under the condition that the rotary valve is positioned at the second valve position, wherein the second reaction comprises the step of enabling the nucleic acid molecules in the reaction device after the first reaction is carried out to interact with the second reaction liquid so as to realize the sequence determination of the nucleic acid molecules.

The method, system, computer-readable storage medium, and computer program product for sequencing nucleic acids according to any of the above embodiments are particularly applicable to a fluidic system involving multi-site three-way control, in which a rotary valve is controlled to independently or concurrently control a first flow channel and a second flow channel, and the number of valve elements to be arranged in a fluid path involving multi-site flow splitting and merging can be effectively reduced, thereby enabling automation and industrialization by easily arranging corresponding hardware structures while enabling various reactions. Generally, a liquid path system or equipment comprising the rotary valve, which can realize the method, has the advantages of low preparation cost, small volume, low reagent consumption, high reliability and convenient maintenance, maintenance and operation; by using the automatic sequencing system comprising any rotary valve or any liquid path system, the flow channel is controlled and selected by enabling the rotary valve to be positioned at different valve positions, so that the flow directions of different liquids can be independently controlled and operated, and the sequence determination of nucleic acid molecules is easier to realize.

Additional aspects and advantages of embodiments of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of embodiments of the present application.

Drawings

The above and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is an exploded schematic view of a rotary valve according to an embodiment of the present application;

fig. 2 is a schematic structural view of a fluid path system according to an embodiment of the present application;

FIG. 3 is a schematic perspective view of a stator and rotor arrangement according to an embodiment of the present application;

FIG. 4 is a schematic illustration of a rotor of an embodiment of the present application in a first valve position;

FIG. 5 is a schematic view of the rotor of an embodiment of the present application in a second valve position;

fig. 6 is a schematic perspective view of a stator according to an embodiment of the present application;

FIG. 7 is a schematic perspective view of a rotor according to an embodiment of the present application;

FIG. 8 is a schematic structural view of the interior of a rotary valve portion of an embodiment of the present application;

FIG. 9 is a schematic plan view of a rotary valve according to an embodiment of the present application;

FIG. 10 is a schematic cross-sectional view of the rotary valve of FIG. 9 taken along A-A;

FIG. 11 is another exploded schematic view of a rotary valve according to an embodiment of the present application;

FIG. 12 is another schematic cross-sectional view of a rotary valve according to an embodiment of the present application;

FIG. 13 is a bottom view schematic of the stator and valve head mating of an embodiment of the present application;

FIG. 14 is a side schematic view of a stator and valve head mating of an embodiment of the present application;

FIG. 15 is a schematic perspective view of a stator according to another embodiment of the present application;

FIG. 16 is a schematic perspective view of a rotor according to another embodiment of the present application;

fig. 17 is another schematic structural view of a fluid path system according to the embodiment of the present application;

fig. 18 is still another schematic structural view of a fluid path system according to an embodiment of the present application;

FIG. 19 is a further schematic structural view of a fluid path system according to an embodiment of the present application;

FIG. 20 is a schematic flow diagram of a sequencing method according to an embodiment of the present application;

FIG. 21 is a schematic block diagram of a nucleic acid sequencing system according to an embodiment of the present application;

FIG. 22 is a schematic diagram of another module of the nucleic acid sequencing system according to the embodiment of the present application;

FIG. 23 is a schematic flow diagram of another sequencing method according to an embodiment of the present application;

FIG. 24 is a further schematic flow diagram of a sequencing method according to an embodiment of the present application.

Description of the main element symbols:

rotary valve 10, fluid path system 12, pump assembly 14, common port 16, first port 18, second port 20, communication channel 21, first communication channel 22, stator 24, rotor 26, second communication channel 28, stator end face 30, rotor end face 32, valve body 34, valve head 36, first interface 38, second interface 40, third interface 42, recess 44, flange 46, top face 48, side face 50, positioning structure 52, end cap 53, positioning pin 54, first fastener 55, drive member 56, second fastener 58, fluid network 60, reaction device 62, memory 64, first memory 66, second memory 68, first reaction device 70, second reaction device 72, first rotary valve 74, second rotary valve 76, first reaction region 78, second reaction region 80, three-way valve 82, multi-way valve 84, liquid inlet 86, liquid outlet 88, liquid collector 89, nucleic acid sequencing system 90, fluid flow control system, and method, A detection assembly 92, a controller 94, a first control module 96, a second control module 98, a third control module 100, a fourth control module 102, a third memory 104, and a fourth memory 106.

Detailed Description

Embodiments of the present application will be further described below with reference to the accompanying drawings. The same or similar reference numbers in the drawings identify the same or similar elements or elements having the same or similar functionality throughout.

In addition, the embodiments of the present application described below in conjunction with the accompanying drawings are exemplary and are only for the purpose of explaining the embodiments of the present application, and are not to be construed as limiting the present application.

In the present application, "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance, order or implied number of the indicated technical features. Thus, features defined as "first", "second", may explicitly or implicitly include one or more of the described features. In the description of the present application, "a plurality" means two or more unless otherwise limited.

In this application, unless expressly stated or limited otherwise, "mounted" and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; may be mechanically connected, may be electrically connected or may be in communication with each other; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art as appropriate.

In this application, unless expressly stated or limited otherwise, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through intervening media. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

In the present application, the terms "nucleic acid sequencing", "sequencing" and "sequencing" are equivalent and include DNA sequencing and/or RNA sequencing, including long fragment sequencing and/or short fragment sequencing; the so-called "sequencing reaction" is the same as the sequencing reaction. Generally, in nucleic acid sequence determination, a base/nucleotide on a template nucleic acid can be identified/determined by one round of sequencing reaction, the base being selected from at least one of A, T, C, G and U. In the sequencing reactions of SBS and/or SBL, the so-called round of sequencing reactions include extension reactions (base extension), signal detection (e.g. photo/image capture) and group excision (excision of detectable and/or blocking groups). The so-called "nucleotide analogs", i.e., substrates, also known as reversible terminators (reversible terminators), are analogs of A, T, C, G and/or U nucleotides that are capable of pairing with a particular type of base following the base complementarity principle, while being capable of blocking or inhibiting the binding of the nucleotide/substrate to the next nucleotide position of the template strand. One round of sequencing reaction comprises one or more repeated reactions consisting of extension reaction (base extension), signal detection and radical excision; for example, four nucleotide analogs carry the same detectable label, e.g., the same fluorescent molecule, and a round of sequencing reactions involves four repetitive reactions, performed sequentially, corresponding to the four nucleotides; for another example, four nucleotide analogs carry two detectable labels separated by a detectable signal region, every two detectable labels are the same, one round of sequencing reaction comprises two repeated reactions, namely, two nucleotides with different detectable labels carry out extension reaction, signal detection and radical excision in the same reaction system, and the two repeated reactions realize the base type identification of one position of the template after one round of sequencing reaction; as another example, four nucleotide analogs each carry four detectable labels with a signal detectable region, or three of the four nucleotide analogs each carry three detectable labels with a signal detectable region, and another nucleotide analog does not carry a detectable label, and a round of sequencing reactions involves subjecting the four nucleotide analogs to a single repetitive reaction in the same reaction system.

Referring to fig. 1-2, the present disclosure provides a rotary valve 10, the rotary valve 10 has a plurality of flow passages, the rotary valve 10 can be applied to a fluid path system 12, or the fluid path system 12 includes the rotary valve 10, and performs merging, splitting, flow path switching and/or flow rate control.

Referring to fig. 3-5, the rotary valve 10 of the present embodiment is configured to rotate between a first valve position and a second valve position, the rotary valve 10 having a common port 16, a plurality of first ports 18, a plurality of second ports 20, and a plurality of communication grooves 21. The communication groove 21 selectively communicates the common port 16 and the first port 18 or the first port 18 and the second port 20.

When the rotary valve 10 is in the first valve position, the first port 18 and the second port 20 communicate with each other through the communication groove 21, and a first flow passage is formed. When the rotary valve 10 is in the second valve position, the first port 18 and the common port 16 communicate with each other through the communication groove 21, and a second flow passage is formed.

In the rotary valve 10 and the fluid passage system 12 according to the embodiment of the present application, each first port 18 communicates with the corresponding second port 20 through the corresponding communication groove 21 when the rotary valve 10 is in the first valve position, and each first port 18 communicates with the common port 16 through the corresponding communication groove 21 when the rotary valve 10 is in the second valve position, so that the rotary valve 10 has two or more passages; the rotary valve 10 is applicable to any fluid path system requiring the use of three-way valves, particularly multiple three-way valves, for example those involving the need to change the direction of one or more flow paths and/or dispense multiple liquids/solutions. The rotary valve can be used for realizing independent or parallel three-way control of a plurality of liquid paths in a liquid path system comprising the plurality of liquid paths/flow paths, and can avoid simultaneously arranging a plurality of three-way valves in the liquid path system 12, so that the cost of the liquid path system 12 is reduced, the size is reduced, the reagent consumption is reduced, the reliability is improved, and the maintenance, the maintenance and the control are convenient.

Specifically, the number of the common ports 16 is one, and the common ports 16 may serve as liquid inlets or outlets of the rotary valve 10. Alternatively, fluid may enter or exit rotary valve 10 from common port 16, thereby achieving either a split flow or a co-current flow. Here, the common port 16 serves as a liquid inlet of the rotary valve 10, and liquid can enter the rotary valve 10 from the common port 16. The shape of the common port 16 may be a regular shape such as a circle, a polygon, or an irregular shape. In the present embodiment, the common port 16 is circular in shape to facilitate forming, manufacturing, and/or connecting the common port 16 to a common conduit.

The plurality of second ports 20 correspond one-to-one to the plurality of first ports 18. The plurality of communication grooves 21 correspond one-to-one to the plurality of first ports 18. The number of first ports 18 may be 2, 3, 4, 5, 6, 7, 8, or more. In the embodiment shown in fig. 6, the number of first ports 18 is 8 to accommodate elements or devices comprising 8 outlets or inlets upstream or downstream of the rotary valve 10. As shown in fig. 2, the liquid enters the rotary valve 10 from the common port 16, flows out from the 8 first ports 18, and then enters the reaction device 62 comprising 8 channels or 8 reaction regions, so that the parallel control of the liquid inlet of the 8 channels or 8 reaction regions of the reaction device 62 can be realized. The shape of the first port 18 may be a regular shape such as a circle, a polygon, or an irregular shape. In the present embodiment, the first port 18 has a circular shape. The first port 18 may be used as a liquid outlet or as an inlet of the rotary valve 10.

Similarly, the number of the second ports 20 is not limited in the present application, and the number of the second ports 20 may be 2, 3, 4, 5, 6, 7, 8 or more. In the embodiment shown in fig. 6, the number of the second ports 20 is 8 to adapt an element or device comprising 8 outlets or inlets upstream or downstream of the rotary valve 10, such as the reaction device 62 comprising 8 channels or 8 reaction zones downstream of the rotary valve 10 in fig. 2, to achieve parallel control of the feeding of the 8 channels or 8 reaction zones of the reaction device 62. The number of second ports 20 is equal to the number of first ports 18. The shape of the second port 20 may be a regular shape such as a circle, a polygon, or an irregular shape. In the present embodiment, the second port 20 has a circular shape. The second port 20 may be used as a liquid inlet or outlet of the rotary valve 10. For ease of manufacture, the second port 20 may be the same size as the first port 18.

The number of the communication grooves 21 is the same as the number of the first ports 18 and/or the second ports 20. In the present embodiment, the number of the communication grooves 21 is also 8. The shape of the communication groove 21 is not limited as long as it allows the first port 18 and the second port 20 to communicate with each other when the rotary valve 10 is in the first valve position, and may be, for example, an arc shape or a rectangular shape.

As shown in fig. 4, in the second valve position, the common port 16 communicates with each of the first ports 18 through a corresponding one of the communication grooves 21, and at this time, the liquid can enter the rotary valve 10 from the common port 16, pass through the communication groove 21, and then flow out from the first port 18. Alternatively, in the second valve position, fluid enters rotary valve 10 from a common port 16 and exits through a plurality of first ports 18. That is, the rotary valve 10 has a single inlet to multiple outlets.

As shown in fig. 5, in the first valve position, each first port 18 is in communication with a corresponding one of the second ports 20, and fluid enters the rotary valve 10 from the first port 18 and exits the corresponding second port 20. That is, the rotary valve 10 also has the function of simultaneously feeding liquid into a plurality of passages and simultaneously discharging liquid from a plurality of passages.

The angle between the first valve position and the second valve position is not specifically limited in the embodiments of the present application. Generally, the angles of the first and second valve positions are related to the distribution and number of positions of the first port 18, the second port 20, and/or the communication groove 21. In one example, the number of the first ports 18, the second ports 20 and the communication grooves 21 is 8, and when the rotary valve 10 is located at the first valve position, the rotary valve 10 can be rotated to the second valve position by rotating the rotary valve 10 clockwise or counterclockwise by 15 degrees.

In the present embodiment, with the rotary valve 10 in the first valve position, both the first port 18 and the second port 20 are blocked from the common port 16; with the rotary valve 10 in the second valve position, the common port 16 and the first port 18 are both blocked from the second port 20 to form separate first or second flow passages. The first flow channel and the second flow channel are multiple, so that parallel control of liquid inlet and outlet of a plurality of reaction areas is facilitated, and independent control of liquid inlet and outlet of different reaction processes on the reaction areas is facilitated.

In some embodiments, the rotary valve 10 may be in a third valve position, in which case the common port 16, the plurality of first ports 18, and the plurality of second ports 20 are isolated from one another. In this manner, the rotary valve 10 may also perform the function of controlling the fluid shutoff with the rotary valve 10 in the third valve position.

In this embodiment, the rotary valve 10 has three modes, i.e., a mode in which the first port 18 and the second port 20 are in communication when the rotary valve 10 is in the first valve position; a mode in which the common port 16 and the first port 18 are in communication when the rotary valve 10 is in the second valve position; a mode in which the common port 16, the first port 18, and the second port 20 are isolated from one another when the rotary valve 10 is in the third valve position.

It should be noted that reference herein to a plurality of ports being "isolated" means that there is no communication between the plurality of ports, or that fluid cannot enter a given port or ports and exit another given port or ports.

Referring to fig. 6 and 7, in some embodiments, the rotary valve 10 includes a stator 24 and a rotor 26 disposed opposite the stator 24, the stator 24 having the common port 16, the plurality of first ports 18, and the plurality of second ports 20. The rotor 26 is provided with at least a part of the plurality of communication grooves 21. As such, during rotation of the rotor 26, the communication groove 21 may communicate the common port 16 and the first port 18, or may communicate the first port 18 and the second port 20, such that liquid may enter the rotary valve 10 from the stator 24 and exit the rotary valve 10 from the stator 24.

Specifically, the rotor 26 and the stator 24 are coaxially arranged, or a central axis of the rotor 26 and a central axis of the stator 24 coincide. It will be appreciated that the rotor 26 may rotate relative to the stator 24. Further, the rotor 26 rotates relative to the stator 24 between the first valve position and the second valve position.

It should be noted that the above-described rotation of the rotary valve 10 between the first valve position and the second valve position means that the rotor 26 of the rotary valve 10 itself rotates and not the rotary valve 10 as a whole.

In addition, the rotor 26 provided with at least a part of the plurality of communication grooves 21 means: in the plurality of communication grooves 21, a part of the number of the communication grooves 21 or the entire data may be provided on the rotor 26, and a part of the structure or the entire structure of the communication grooves 21 may be provided on the rotor 26.

In the present embodiment, the communication groove 21 may have a bent shape, a curved shape, or the like, and the specific shape of the communication groove 21 is not limited herein.

In some embodiments, the communication groove 21 includes a first communication groove 22 and a second communication groove 28 that are communicable, and the first communication groove 22 and the second communication groove 28 are provided in the rotor 26 and the stator 24, respectively. Thus, the communication groove 21 is more easily formed.

In the present embodiment, the plurality of first connecting grooves 22 are arranged at intervals in the circumferential direction of the rotor 26. The plurality of second communication grooves 28 are radially arranged with the common port 16 as a center.

In other embodiments, the first connecting groove 22 may be omitted, and in this case, the connecting groove 21 includes the second connecting groove 28, and since the second connecting groove 28 is provided in the stator 24, the entire structure of the connecting groove 21 is provided in the stator 24, or the stator 24 is provided with the connecting groove 21. In this case, the first port 18 and the second port 20 may both be provided on the rotor 26 and the common port 16 on the stator 24. The communication groove 21 may be provided as a first portion and a second portion, and the first portion and the second portion may be provided in a partitioned manner. One end of the first portion may be in communication with the common port 16 and the other end may be in selective communication with the first port 18 during rotation of the rotor 26. The second portion selectively communicates with the first and

second ports

18, 20 during rotation of the rotor 26, thereby placing the first and

second ports

18, 20 in communication.

Of course, similarly, the second communication groove 28 may be omitted, and in this case, the communication groove 21 includes the first communication groove 22, and since the first communication groove 22 is provided on the rotor 26, the entire structure of the communication groove 21 is provided on the rotor 26, or in other words, the rotor 26 is provided with the communication groove 21. In this case, the first port 18, the second port 20, and the common port 16 may be provided in the stator 24, and the communication groove 21 may be provided in a first portion and a second portion, the first portion being provided in a blocked manner from the second portion. One end of the first portion may be in communication with the common port 16 and the other end may be in selective communication with the first port 18 during rotation of the rotor 26. The second portion selectively communicates with the first and

second ports

18, 20 during rotation of the rotor 26, thereby placing the first and

second ports

18, 20 in communication.

In some embodiments, one communication groove 21 is formed by one first communication groove 22 and one second communication groove 28, the one first communication groove 22 has two ends, one end of each second communication groove 28 communicates with one first port 18 and the other end selectively communicates with one second port 20, the one second communication groove 28 has two ends, and one end of each second communication groove 28 communicates with the common port 16 and the other end selectively communicates with one first port 16. In this way, the second communicating groove 28 is designed to allow the common port 16 to be easily communicated with the first port 18 by rotating the rotary valve 10, and further, the communicating groove 21 including the first communicating groove 22 and the second communicating groove 28 can facilitate control of the rotary valve 10, so that rotation of the rotary valve 10 can realize both the above-mentioned designated functions and the preparation.

Specifically, the number of the flow passages 28 is the same as the number of the first ports 18, and in the present embodiment, the number of the second communication grooves 28 is 8. Of course, in other embodiments, the number of flow channels 28 may be 2, 3, 4, 5, 6, and other numbers.

Referring to fig. 6 and 7, in some embodiments, the stator 24 includes a stator end 30, the rotor 26 includes a rotor end 32, and the stator end 30 is attached to the rotor end 32. The second communication groove 28 is formed in the stator end surface 30. The first communicating groove 22 is formed in the rotor end face 32.

Thus, the rotor end face 32 and the stator end face 30 are attached to prevent liquid leakage, the second communicating groove 28 is formed in the stator end face 30, and the first communicating groove 22 is formed in the rotor end face 32, so that the second communicating groove 28 and the first communicating groove 22 are easier to manufacture and form, and the manufacturing cost of the stator 24 and the rotor 26 can be reduced.

In certain embodiments, both the plurality of first ports 18 and the plurality of second ports 20 are disposed around the common port 16. In this manner, liquid entering the common port 16 may flow around to exit the first port 18, facilitating a more uniform flow of liquid. Additionally, the second port 20 surrounding the common port 16 may prevent the second port 20 from interfering with the common port 16.

In certain embodiments, the plurality of first ports 18 are spaced circumferentially along the stator 24 or rotor 26. Thus, the arrangement of the first ports 18 can reasonably utilize the space of the stator 24, and the concentration of the first ports 18 is avoided, so that the volume of the stator 24 can be reduced. Further, the plurality of first ports 18 are uniformly spaced along the circumferential direction of the stator 24, or, along the circumferential direction of the stator 24, the intervals between two adjacent first ports 18 are equal.

In certain embodiments, the plurality of second ports 20 are spaced circumferentially along the stator 24 or rotor 26. Thus, the second ports 20 are arranged in a manner that the space of the stator 24 can be reasonably utilized, and the concentration of the second ports 20 is avoided, so that the volume of the stator 24 can be reduced. Further, the plurality of second ports 20 are uniformly spaced along the circumferential direction of the stator 24, or, along the circumferential direction of the stator 24, the intervals between two adjacent second ports 20 are equal.

As shown in fig. 6, in certain embodiments, the plurality of first ports 18 and the plurality of second ports 20 are located on the same circumference. Alternatively, the plurality of first ports 18 and the plurality of second ports 20 are arranged in a circular ring shape. In this way, the first port 18 and the second port 20 are more easily opened. Further, the alternating arrangement of the first port 18 and the second port 20 may make it easier for the rotary valve 10 to switch from the first valve position to the second valve position during rotation of the rotor 26.

In some embodiments, the rotary valve 10 has a central axis, and the plurality of first ports 18 are spaced apart on a circular plane having the central axis of the rotary valve 10 as a central axis. In this way, the first port 18 and the second port 20 are more easily opened.

Further, the plurality of second ports 20 are arranged at intervals on a circular plane having the central axis of the rotary valve 10 as the axis.

Referring to FIG. 1, in some embodiments, the rotary valve 10 includes a valve body 34, and the stator 24 and the rotor 26 are housed within the valve body 34. In this manner, the valve body 34 acts as a carrier for the rotary valve 10, and the valve body 34 may facilitate installation of the stator 24, the rotor 26, and other components of the rotary valve 10.

Specifically, the valve body 34 may be made of metal or the like, so that the strength of the valve body 34 may be increased, so that the valve body 34 may protect the rotor 26 and the stator 24, thereby increasing the lifespan of the rotary valve 10. The valve body 34 is in a cylindrical shape, a rectangular parallelepiped shape, or the like, and the valve body 34 may be designed in different shapes according to actual requirements, without limiting the shape of the valve body 34.

Referring to fig. 1 and 8, in some embodiments, the rotary valve 10 includes a valve head 36, the valve head 36 covers the stator 24 and is disposed on the valve body 34, the valve head 36 has a first port 38, a second port 40, and a third port 42, the first port 38 is in communication with the common port 16, the second port 40 is in communication with the first port 18, and the third port 42 is in communication with the second port 20.

In this manner, the interface of the valve head 36 may be connected to external piping so that the rotary valve 10 may control the dispensing of liquid. Specifically, referring to fig. 9 and 10, the edge of the valve body 34 has a groove 44, and the edge of the valve head 36 has a flange 46, and the flange 46 is clamped in the groove 44 to make the valve head 36 and the valve body 34 fit more compactly.

The first port 38 may be a threaded port or an external conduit or the like may be threadably coupled to the rotary valve 10. The threaded interface not only prevents fluid from leaking through the first interface 38, but also allows the external conduit to be quickly removed from the rotary valve 10. Similarly, the second port 40 and the third port 42 may also be threaded ports.

The valve head 36 is a cap-like member, and the valve head 36 includes a top surface 48 and a side surface 50 connecting the top surface 48, the side surface 50 being disposed obliquely with respect to the top surface 48. The first port 38 and the second port 40 are each disposed on the top surface 48 and the third port 42 is disposed on the side surface 50. Therefore, the positions of the interfaces are dispersed, and mutual interference among the interfaces is avoided.

Referring to FIG. 8, in certain embodiments, the rotary valve 10 includes positioning structure 52, the positioning structure 52 being used to position the valve head 36 and the stator 24. In this manner, the locating structure 52 allows for more accurate positioning between the valve head 36 and the stator 24, such that the ports and corresponding ports on the valve head 36 may communicate, facilitating fluid flow.

Specifically, in one example, the locating structure 52 includes a locating pin 54, the locating pin 54 being interposed in the valve head 36 and the stator 24. In the embodiment of the present application, the number of the positioning pins 54 is plural, and the plural positioning pins 54 can limit the degree of freedom of the valve head 36 and the stator 24, so that the valve head 36 is positioned on the stator 24.

In certain embodiments, as shown in fig. 1 and 10, the rotary valve 10 further includes an end cap 53 and a first fastener 55, and the valve head 36 is secured to the valve body 34 by the end cap 53 and the first fastener 55. The end cap 53 may be in the form of an annular plate, and the first fastening member 55 may be a screw.

In one example, the rotary valve 10 is assembled by first installing the stator 24 and the rotor 26 into the valve body 34, then installing the valve head 36 on the valve body 34 and positioning the valve head 36 and the stator 24, then placing the end cap 53 over the valve head 36, and finally threading the end cap 53 and the valve head 36 through the first fastener 55 and into the valve body 34 such that the valve head 36 is secured to the valve body 34.

Referring to fig. 11-14, in some embodiments, the valve head 46 and the stator 24 may be a unitary structure. Alternatively, the valve head 46 and stator 24 may not be removable. For example, the valve head 46 and the stator 24 may be formed by removing material from the same substrate. In such embodiments, the positioning structure 52 may be omitted. Alternatively, the valve head and stator need not be mounted together using the locating structure 52.

It should be noted that for further explanation of the rotary valve of the embodiment of fig. 11-14, reference is made to the same or similar parts of the rotary valve of the embodiment of fig. 1-10, which are not repeated herein.

Referring again to fig. 1, in some embodiments, the rotary valve 10 includes a driving member 56 coupled to the rotor 26, the driving member 56 is disposed on the valve body 34, and the driving member 56 is configured to drive the rotor 26 to rotate. In this manner, the drive member 56 may drive the rotor 26 to rotate such that the rotor 26 may be positioned at different locations to enable different functions of the rotary valve 10.

In particular, the drive member 56 may be a motor, the rotating portion of which is coupled to the rotor 26. The drive member 56 may be secured to the valve body 34 by a second fastener 58 such that the relative position between the drive member 56 and the valve body 34 is fixed.

Referring to fig. 15-16, fig. 15-16 are perspective views of a stator 204 and a rotor 206, respectively, according to another embodiment of the present application. In this embodiment, the rotor 206 has the common port 106, the plurality of first ports 108, and the plurality of second ports 200, and the stator 204 is provided with at least a part of the plurality of communication grooves 202. In other words, the common port 106, the first port 108, and the second port 200 are formed in the rotor 206, and at least a part of the communication grooves 202 of the plurality of communication grooves 202 is formed in the stator 204. In this embodiment, other explanations of the common port 106, the first port 108 and the second port 200 may refer to the explanations of the stator 24 and the rotor 26 of any of the above embodiments, and are not repeated herein. For example, the stator 204 provided with at least a part of the plurality of communication grooves 202 means: in the plurality of communication grooves 202, a part of the number of the communication grooves 202 or the entire data may be provided on the stator 204, and a part of the structure or the entire structure of the communication grooves 202 may be provided on the stator 204.

Referring again to fig. 2, the fluid circuit system 12 of one embodiment of the present application includes the rotary valve 10 of any of the above embodiments, a pump assembly 14, and a fluid network 60, the fluid network 60 including a reservoir 64, a first flow channel, a second flow channel, and a reaction device 62, the first flow channel and the second flow channel each independently fluidly connecting the reservoir 64 and the reaction device 62, the pump assembly 14 being in communication with the rotary valve 10. The pump assembly 14 is in communication with a fluid network 60. The rotary valve 10 is configured to rotate between a first valve position, in which the pump assembly 14 induces the flow of the liquid in the reservoir 64 toward the reaction device 62 through the first flow channel, and a second valve position, in which the rotary valve 10 is in the second valve position, in which the pump assembly 14 induces the flow of the liquid in the reservoir 64 toward the reaction device 62 through the second flow channel.

In the fluid path system 12 of the embodiment of the present application, the rotary valve 10 can be used to independently or parallelly control the first flow channel and the three-way valve of the first flow channel, and it can be avoided that a plurality of three-way valves are simultaneously arranged in the fluid path system 12, so that the cost of the fluid path system 12 is reduced, the volume is reduced, the reagent consumption is reduced, the reliability is improved, and the maintenance, the maintenance and the control are convenient. The fluid path system 12 described above is particularly suited for use in systems requiring high precision in fluid control and delivery, such as sequencing systems.

Specifically, the pump assembly 14 may power the fluid circuit system 1212 so that fluid may flow. The pump assembly 14 may contain a plurality of pumps, such as two, where one pump may be in communication with the common port 16 and the other pump may be in communication with the second port 20, either in split or in parallel flow. A pump in communication with the common port 16 may provide power to the liquid from the common port 16 to the first port 18, and a pump in communication with the second port 20 may provide power to the liquid from the second port 20 to the first port 18.

In one embodiment, the number of the first ports 18 is 8, and the pump assembly 14 comprises 8 pumps equal to the number of the first ports 18, such as a negative pressure octal pump, which is located downstream of the fluid path system 1212, specifically, downstream of the reaction device 62, and which can independently provide negative pressure to the fluid passing through the rotary valve 10 in an eight-in first flow channel or an eight-in eight-out second flow channel, so that the power level of the fluid in each flow channel of the fluid network 60 can be independently controlled, thereby facilitating fine control of the flow rate and/or velocity of the fluid in each flow channel.

The reservoir 64 may store a plurality of solutions, including reaction solutions, buffers, wash solutions, and/or purified water, etc., including reagents for different reactions or for different steps of a reaction, and the fluid circuit 12 including the pump assembly 14 may allow one or more solutions to flow toward the reaction device 62 sequentially or simultaneously.

In certain embodiments, the reservoir 64 includes a first reservoir 66 and a second reservoir 68, the first reservoir 66 carrying the biological sample solution and the second reservoir 68 carrying the reaction solution. With the rotary valve 10 in the first valve position, the rotary valve 10 communicates the first reservoir 66 and the reaction device 62, and the pump assembly 14 induces the flow of the biological sample solution through the first flow channel toward the reaction device 62. With the rotary valve 10 in the second valve position, the rotary valve 10 communicates the second reservoir 68 with the reaction device 62, and the pump assembly 14 induces the reaction fluid to flow through the second flow path toward the reaction device 62.

That is, the rotary valve 10 can allow the reaction solution and the biological sample solution to enter the reaction device 62 independently and separately in time to sequentially perform corresponding reactions, such as sample loading reaction (nucleic acid immobilization and/or hybridization) and sequencing reaction.

Illustratively, the first reaction is, for example, a fixation and/or hybridization reaction, i.e., a nucleic acid to be tested is fixed or attached to a channel or reaction area of the reaction device 62, and the biological sample solution is a solution containing the nucleic acid to be tested, and the biological sample solution may sequentially enter the reaction device 62 through the second port 20 and the first port 18 to perform the first reaction. In one example, the reaction device 62 is a sandwich-like structure having three layers, or a structure with an upper layer (close to the objective lens) and a lower layer, wherein the upper layer (close to the objective lens) is a transparent glass layer, the middle layer or the lower layer is a transparent glass layer or an opaque substrate layer, the middle layer or the lower layer is provided with a plurality of channels arranged in an array, the channels can accommodate liquid to provide physical space for reaction, each channel is provided with an independent liquid inlet and an independent liquid outlet, the number of the channels is equal to that of the first ports 18, a plurality of biological sample solutions can simultaneously and independently enter one channel of the reaction device 62 through one second port 20 and one first port 18, thus, the rotary valve 10 or the fluid path system 12 including the rotary valve 10 can perform a loading and detection analysis of multiple biological samples without the need to combine other means (e.g., labeling different biological samples). Specifically, the reaction device 62 is, for example, a solid phase substrate having a functional group on the surface and/or a solid phase substrate having a probe attached to the surface, the solid phase substrate having a functional group on the surface and/or the solid phase substrate having a probe attached to the surface are also generally referred to as a chip or a microsphere, for example, the channel of the upper surface (upper glass layer of the lower surface) and/or the channel of the lower surface (middle or lower structure of the upper surface) has a functional group or connected with a probe (oligonucleotide), the functional group can be connected with the nucleic acid to be detected, and/or the probe at least a portion can be complementary with the nucleic acid to be detected and paired, to immobilize or attach the nucleic acid to be detected to the surface of the solid phase substrate, for performing a subsequent detection analysis, such as a sequencing reaction, on the nucleic acid to be detected immobilized or attached to the surface of the solid phase substrate.

The second reaction is, for example, a sequencing reaction, i.e., a nucleic acid sequencing reaction, more specifically, a sequencing reaction while synthesis using a reversible terminator based on chip detection, and accordingly, the reaction solution includes one or more reagents including a substrate (reversible terminator), a polymerase catalyst, a cleavage reagent (group excision reagent), an imaging reagent, and a washing reagent, and the reagents/reaction solutions may sequentially or simultaneously enter the reaction device 62 through the common port 16 and the first port 18 to perform the second reaction, and specifically, the reagents may sequentially or simultaneously flow to the reaction device 62 after passing through the rotary valve 10 to perform a plurality of reaction steps in the reaction device 62 to achieve the sequencing reaction; the reaction device 62 is, for example, a solid substrate having a nucleic acid to be detected attached to a surface thereof, and the solid substrate having the nucleic acid to be detected attached to the surface thereof is, for example, a chip or a microsphere.

The above-described first reaction and second reaction can be carried out in the reaction apparatus 62 by controlling one rotary valve 10 to switch the flow path, so that the nucleic acid sequencing system (integrated system) including the first reaction and the second reaction can be made to have a simpler structure. Thus, the first reaction and the second reaction do not need to be carried out in different systems/devices/apparatuses, the operation of a user is simpler, and the cost for constructing the integrated nucleic acid sequence measuring system is far lower than the sum of the cost for constructing a nucleic acid sequence measuring system (sample loading device) for separately realizing the first reaction and the cost for separately realizing the second reaction.

The first reaction of the present embodiment includes a reaction for attaching a biomolecule to the reaction device 62, and includes, for example, an immobilization, hybridization, or sampling reaction. The term biomolecule includes DNA and/or RNA and the like, including ribonucleotides, deoxyribonucleotides and analogs thereof, including A, T, C, G and U and analogs thereof. Wherein C represents cytosine or a cytosine analogue, G represents guanine or a guanine analogue, A represents adenine or an adenine analogue, T represents thymine or a thymine analogue, and U represents uracil or a uracil analogue. The reaction device 62 is referred to as a reaction chamber, the reaction device 62 may be a chip, and the reaction device 62 is detachably connected to the liquid path system 12.

The second reaction includes a reaction for detecting a biomolecule, for example, a nucleic acid, attached to the reaction device 62, and the second reaction may be a sequencing reaction, so-called sequencing, including determining the primary structure or sequence of DNA or RNA, etc., including determining the order of nucleotides/bases of a given nucleic acid fragment. The second reaction may include one or more sub-reactions. In one example, sequencing the DNA, the second reaction is sequencing, sequencing by synthesis or sequencing by ligation, in particular, sequencing by synthesis, for example, based on chip detection, using a modified nucleotide with a detectable label, such as a dNTP or a dNTP analogue with a detectable label, the sequencing comprising a plurality of sub-reactions, including a base extension reaction, signal acquisition and detection group excision, to effect determination of the base type at a position on the nucleic acid sequence to be detected; performing the plurality of sub-reactions once may be referred to as performing one repeat reaction or one round of reaction, and sequencing comprises performing a plurality of repeat reactions or multiple rounds of reactions to determine the nucleotide/base order of at least one sequence of the nucleic acid molecule (template). The modified nucleotide is said to carry a fluorescent molecule which in a particular context is capable of being excited to fluoresce for detection by an optical system, and when bound to the test nucleic acid, the modified nucleotide label prevents base/nucleotide binding to the next position in the test nucleic acid, for example a dNTP having a chemically cleavable moiety at the 3' hydroxyl terminus or a dNTP having a molecular conformation which prevents the binding of the next nucleotide to the test nucleic acid, the dNTP or dNTP analogue being four deoxyribonucleotides comprising bases A, T/U, C and G respectively.

For sequencing-by-synthesis (SBS) or sequencing-by-ligation (SBL) based on chip detection, under the action of polymerase or ligase, the base extension reaction involves binding of nucleotide glycosides (including modified nucleotides) to the nucleic acid molecules to be detected based on the base complementation principle on a reaction device 62 in which the nucleic acid molecules to be detected are immobilized, and collecting the corresponding reaction signals. The modified nucleotide may be a nucleotide with a detectable label that allows the modified nucleotide to be detected under certain circumstances, for example, a nucleotide with a fluorescent molecular label that fluoresces when excited by a laser of a particular wavelength; typically, for SBS, the engineered nucleotide also has the function of inhibiting the binding of another nucleotide to the next position of the same nucleic acid molecule, e.g. with a blocking group that prevents the binding of other nucleotides to the next position of the template, so that each extension reaction is a single base extension reaction to enable the acquisition of the corresponding signal from a single secondary base extension, the blocking group being e.g. an engineered azide (-N) attached at the 3' position of the sugar residue of the nucleotide₃)。

For the detection and analysis of the biomolecules, generally, the biomolecules are connected to the reaction device 62, and then the biomolecules connected to the reaction device 62 are detected; specifically, in any of the above embodiments, the first reaction is performed before the second reaction, i.e., the sequencing reaction is performed after the nucleic acid to be detected is connected to the reaction device 62. Thus, by using the fluid path system 12, it is possible to perform a plurality of types of reactions including sampling and sample detection in one nucleic acid sequence measurement system.

In some embodiments, the first reaction is a sampling reaction, the second reaction is a sequencing reaction, the first reaction is performed before the second reaction, and the nucleic acid molecule to be detected is contained in a micro-biological sample solution, for example, on a microliter scale, for example, 20 microliters; before the first reaction, the liquid path system 12 is cleaned by using a cleaning solution, for example, a solution that does not affect the subsequent reaction, and the liquid path system 12 is filled with the cleaning solution; before the first reaction is started, a section of air is firstly injected to separate a biological sample solution flowing in subsequently and a cleaning solution in a liquid path system so as to prevent a trace biological sample from being diffused and/or diluted to influence the connection of a nucleic acid molecule to be detected to the reaction device 62 and the subsequent detection of the nucleic acid molecule to be detected, and the separation of the biological sample solution flowing in subsequently and the cleaning solution in the liquid path system is also favorable for observing the sample injection condition, and is favorable for observing whether the reaction device 62, such as a chip comprising a plurality of channels, is normal or not, the liquid path system 12 is normal or not, and the like.

The substrate can be any solid support useful for immobilizing nucleic acid sequences, such as nylon membranes, glass sheets, plastics, silicon wafers, magnetic beads, and the like. Probes can be randomly distributed on the surface of the substrate, can be a section of DNA and/or RNA sequence and the like, and can also be called as a primer, a capture chain or a fixed chain. The first reaction may fixedly attach the biomolecule to the probe, for example, based on the base complementary principle, so that the biomolecule is attached to the reaction device 62.

Acquiring the signal comprises acquiring a signal emitted from the modified nucleotide bound to the nucleic acid molecule, for example, by irradiating a specific region in the reaction device 62 after the base extension reaction with laser light using an optical imaging assembly/system, the fluorescent molecular marker in the specific region being excited to emit fluorescence, and then photographing/image-acquiring the region to record the biochemical reaction signal as image information. The sequencing in turn comprises converting the image information obtained from the multiple rounds/repeats of the reaction into sequence information, i.e.determining the base type based on the image information, so-called base-calling.

Group excision involves removal of the detectable label and/or blocking group bound to the engineered nucleotide of the nucleic acid molecule after the base extension reaction to enable the binding of other nucleotides (including engineered nucleotides) to the next position of the nucleic acid molecule for the next repeat reaction or round of reaction.

A cleaning reagent may also be introduced to remove residual unreacted materials, materials that interfere with the reaction or signal acquisition in the reaction device 62 or in the fluid path system 12 after the previous round or previous sub-reaction or previous step is completed and before the next round or subsequent sub-reaction or next step is started.

In certain embodiments, rotary valve 10 is disposed upstream of reaction device 62. Thus, the rotary valve 10 can control the solution to enter the reaction device 62, and the liquid path system 12 can control the entering and exiting of various solutions to realize various reactions only by using one power assembly (e.g., a pump assembly) or only providing power in one direction, which is beneficial to further reducing the volume of the liquid path system 12 and improving the integration degree thereof, and is beneficial to industrialization.

In certain embodiments, the pump assembly 14 is disposed downstream of the reaction device 62, providing a negative pressure. In this manner, the pump assembly 14 may create a negative pressure in the reaction device 62 and the components of the rotary valve 10, thereby allowing the solution to enter the reaction device 62. In addition, the negative pressure created by the pump assembly 14 can remove air from the reaction device 62, thereby preventing air from affecting the normal reaction of the reaction device 62. The fluid path system 12 including the pump assembly 14 located downstream of the reaction device 62 can provide a uniform power direction for the inlet and outlet of a plurality of types of reactions, and is particularly suitable for the fluid path system 12 including pressure-sensitive elements/components, for example, the reaction device 62 is a chip including thin glass and a multi-layer sheet structure bonded by glue, the chip may further include independent reaction regions/channels, and the change of the power/pressure direction is easy to deform the chip or generate liquid leakage or cross-channel phenomenon.

Referring to FIG. 17, in some embodiments, the reaction apparatus 62 includes a first reaction apparatus 70 and a second reaction apparatus 72, the rotary valve 10 includes a first rotary valve 74 and a second rotary valve 76, and the first rotary valve 74 and the second rotary valve 76 are in communication with the first reaction apparatus 70 and the second reaction apparatus 72, respectively. In this way, the first rotary valve 74 and the second rotary valve 76 can independently control the reactions of the first reaction device 70 and the second reaction device 72, which is beneficial to the first reaction device 70 and the second reaction device 72 to alternately perform different reactions or different steps/sub-reactions of the same reaction, thereby improving the reaction efficiency. In addition, the fluid path system 12 including the rotary valve 10, in combination with the plurality of reaction devices 62 or the reaction devices 62 having a plurality of independent reaction regions, facilitates an increase in the detection throughput and/or enables a plurality of samples to be detected at one time.

Specifically, the first reaction device 70 and the second reaction device 72 may be of a separate structure or an integrated structure. In the example shown in fig. 17, the first reaction device 70 and the second reaction device 72 are of unitary construction. The first reaction device 70 and/or the second reaction device 72 may include one or more reaction zones. Wherein each reaction zone can effect a reaction, the reaction zones can be intersecting, continuous or separate zones.

As in the example of fig. 17, the first reaction device 70 includes eight first reaction zones 78. The eight first reaction zones 78 correspond one-to-one to the eight first ports 18 of the first rotary valve 74. Eight first reaction zones 78 are separately provided. Similarly, the second reaction device 72 includes eight second reaction zones 80. Eight second reaction zones 80 are in one-to-one correspondence with the eight second ports of the second rotary valve 76. Eight second reaction regions 80 are separately provided.

It should be noted that the first rotary valve 74 and the second rotary valve 76 may be operated simultaneously, individually or alternatively, so that the reactions or steps in the first reaction device 70 and the second reaction device 72 may be performed alternately in time-sharing manner or simultaneously, which is beneficial to improving the reaction efficiency and saving the consumption of the detection reagent and/or time.

Referring to fig. 18, in some embodiments, the fluid path system 12 further includes a three-way valve 82 disposed between the reservoir 64 and the rotary valve 10, the three-way valve 82 configured to switch between a first position and a second position, the three-way valve 82 communicating the reservoir 64 and the first rotary valve 74 with the three-way valve 82 in the first position, and the three-way valve 82 communicating the second rotary valve 76 with the three-way valve 82 in the second position. In this way, the three-way valve 82 realizes the liquid inlet time-sharing control of the first rotary valve 74 and the second rotary valve 76, thereby realizing the staggered control of different types of reactions or different steps of the same type of reactions of the first reaction device 70 and the second reaction device 72, and being beneficial to improving the reaction efficiency and saving the time.

For example, one of the first reaction apparatus 70 and the second reaction apparatus 72 performs a first reaction, and the other performs a second reaction. As another example, one of the first reaction apparatus 70 and the second reaction apparatus 72 performs a certain step in the second reaction, and the other performs another step in the second reaction.

Further, in certain embodiments, the three-way valve 82 communicates the common port of the accumulator 64 and the first rotary valve 74 with the three-way valve 82 in the first position, and the three-way valve 82 communicates the common port of the accumulator 64 and the second rotary valve 76 with the three-way valve 82 in the second position.

In this manner, the liquid passing through the three-way valve 82 may enter the reaction device 62 through the second flow path formed by the first rotary valve 74 or the second flow path formed by the second rotary valve 76 to perform a corresponding reaction. For example, the liquid passing through the three-way valve 82 enters the first reaction device 70 through the second flow path of the first rotary valve 74 to perform the second reaction (sequencing reaction).

Referring to fig. 19, in some embodiments, fluid pathway system 12 includes a multi-way valve 84, where multi-way valve 84 has a plurality of fluid inlets 86 and a fluid outlet 88, where fluid outlet 88 is selectively connected to one of fluid inlets 86, and fluid inlet 86 is connected to three-way valve 82. In this manner, the plurality of liquid inlets 86 of the multi-way valve 84 allow the first reaction device 70 or the second reaction device 72 to enter different liquids, thereby achieving multiple rounds/repetitions of reaction.

For example, in the case that the three-way valve 82 communicates the second flow channel of the first rotary valve 74 with the multi-way valve 84, the first reaction device 70 may implement the second reaction, and at this time, when one of the liquid inlet 86 and the liquid outlet 88 is switched to communicate, switching of multiple reagents may be implemented, so as to implement a specified process or step of the second reaction in the second reaction device 72, and through switching of the communication position of the multi-way valve 84, multiple reagents passing through different liquid inlet 86 may enter the first reaction device 70 in a specified order, so as to implement the second reaction.

It can be understood that in the case where the three-way valve 82 communicates the second flow path of the second rotary valve 76 with the multi-way valve 84, the reagent passing through the multi-way valve 84 and the three-way valve 82 may enter the second reaction device 72 to perform the second reaction.

As shown in fig. 19, in certain embodiments, the fluid path system 12 includes a liquid trap 89, and the liquid trap 89 collects liquid flowing out of the reaction device 62. For example, the liquid trap 89 collects the liquid after the first reaction and the second reaction are performed.

Referring to fig. 20, the present application also provides a method for determining a nucleic acid sequence, comprising: s110, rotating the rotary valve 10 provided with the first flow channel and the second flow channel to a first valve position to communicate the first storage 66 with the reaction device 62, wherein the first storage 66 carries a first reaction liquid, and the first reaction liquid contains nucleic acid molecules; s120, under the condition that the rotary valve 10 is at the first valve position, enabling a first reaction liquid to enter the reaction device 62 through the first flow channel to perform a first reaction, wherein the first reaction comprises connecting at least one part of nucleic acid molecules to the reaction device 62; s130, rotating the rotary valve 10 to a second valve position to communicate the second storage 68 and the reaction device 62, wherein the second storage 68 carries a second reaction solution, and the second reaction solution contains components required for nucleic acid sequencing; s140, under the condition that the rotary valve 10 is at the second valve position, a second reaction liquid enters the reaction device 62 through the second flow channel to carry out a second reaction, and the second reaction comprises the steps of enabling the nucleic acid molecules in the reaction device 62 after the first reaction is carried out to interact with the second reaction liquid to carry out a polymerization reaction and detecting a signal from the reaction, so as to realize the sequence determination of the nucleic acid molecules. The method realizes the first reaction and the second reaction by enabling one rotary valve 10 to be in different valve positions to realize the switching of different flow channels/reagents, and is particularly suitable for the operation control of a system or equipment with high integration.

Current generation high throughput sequencing platforms, or single molecule sequencing platforms, generally require processing a sample to be tested before on-line sequencing, such as adapting to a designated sequencing platform, processing the sample to be tested to convert it into a library adapted to the sequencing platform, and loading the library into a designated area, such as the reaction device 62, so as to place the reaction device 62 containing the sample to be tested into a sequencer for automated sequencing. Currently commercially available sequencing platforms, the processing of the sample to be tested before being loaded on the machine is generally separated from the on-machine sequencing, for example, by manually performing sample processing/library preparation in a reagent tube, or by performing the processing and loading of the sample to be tested on a sample processing device. The method can realize sample processing and sequencing before the computer is operated, realize the switching and the in-and-out control of different flow channels of a plurality of reagents by enabling the rotary valve 10 provided with the first flow channel and the second flow channel to be in different valve positions, and is particularly suitable for an integrated sequencing platform integrating the sample processing and sequencing functions before the computer is operated.

Referring to fig. 21, the method for determining a nucleic acid sequence can be implemented by the nucleic acid sequence determination system 90, and specifically, the nucleic acid sequence determination system 90 can include a first control module 96, a second control module 98, a third control module 100 and a fourth control module 102, step S110 can be implemented by the first control module 96, step S120 can be implemented by the second control module 98, step S130 can be implemented by the third control module 100, and step S140 can be implemented by the fourth control module 102.

Alternatively, the first control module 96 is configured to rotate the rotary valve 10 having the first flow channel and the second flow channel to the first valve position to communicate the first reservoir 66 and the reaction device 62, wherein the first reservoir 66 carries a first reaction solution, and the first reaction solution comprises nucleic acid molecules. The second control module 98 is configured to cause the first reaction solution to enter the reaction device 62 through the first flow channel to perform a first reaction with the rotary valve 10 in the first valve position, the first reaction including coupling at least a portion of the nucleic acid molecules to the reaction device 62. The third control module 100 is used for rotating the rotary valve 10 to the second valve position to communicate the second storage 68 and the reaction device 62, wherein the second storage 68 carries a second reaction solution containing components required for nucleic acid sequencing. The fourth control module 102 is configured to enable a second reaction solution to enter the reaction device 62 through the second flow channel to perform a second reaction with the rotary valve 10 in the second valve position, wherein the second reaction comprises interacting the nucleic acid molecule in the reaction device 62 after performing the first reaction with the second reaction solution to perform the sequencing of the nucleic acid molecule.

The first through

fourth control modules

96, 98, 100 and 102 are each independently physical devices such as automated test equipment and/or non-physical devices such as computer executable programs, each independently capable of controlling the operation of components or assemblies such as rotary valves, pump assemblies, and test assemblies, and may be separate devices or modules of a system, which may be located in the same space or in different spaces, which may be physical or digital spaces, but may also be capable of communicating signals or data therebetween.

In the method for determining a nucleic acid sequence and the nucleic acid sequence determination system 90, the first flow channel and the second flow channel can be independently or parallelly three-way controlled by the rotary valve 10, and a plurality of three-way valves can be avoided being arranged in the liquid path system at the same time, so that the cost of the liquid path system is reduced, the volume is reduced, the reagent consumption is reduced, the reliability is improved, and the maintenance, the maintenance and the operation are convenient. In addition, the rotary valve 10 is located at different positions to independently control and operate the flow direction of different liquids, so that the sequence determination of nucleic acid molecules is easier to realize.

Referring to fig. 22, in some embodiments, the method for determining a nucleic acid sequence further comprises performing the following after performing step S120 and before performing step S140 (after performing the first reaction and before performing the second reaction): the third reaction solution in the third storage 104 is made to enter the reaction device 62 through the first flow path or the second flow path to perform a third reaction, the third reaction includes the interaction of the nucleic acid molecule in the reaction device 62 after the first reaction is performed and a third reaction solution to realize the amplification of the nucleic acid molecule, and the third reaction solution contains the components required for the amplification.

The term "amplification" refers to cloning a nucleic acid molecule, for example, by replicating thousands or even millions of copies of the nucleic acid molecule into a cluster (cluster) by polymerase chain reaction, any one of the thousands or millions of copies/cluster being identical in sequence to the original nucleic acid molecule, such that the signal from the nucleic acid molecule can be amplified by increasing the number of molecules, facilitating detection of the nucleic acid molecule; in particular, in subsequent sequencing, the signal emitted by the tens of thousands of millions of molecules (clusters) is equivalent to the signal from the single nucleic acid molecule, greatly enhancing the signal of the molecule, facilitating detection.

Second generation high throughput sequencing platforms currently on the market, such as ILLUMINA sequencing platform, Ion Torrent sequencing platform and chinese geno sequencing platform, require amplification of the signal from the molecule to be detected by amplification, such as bridge PCR, prior to sequencing, to obtain a stronger signal that is easily recognized for detection (or not susceptible to interference).

Referring to fig. 22, in some embodiments, the method for determining a nucleic acid sequence further comprises performing, before performing step S120 (before performing the first reaction): the washing solution in the fourth reservoir 106 is introduced into the reaction device 62 through the first flow path. Thus, the washing solution can rinse the reaction device 62, thereby preventing the first reaction solution from being contaminated by the last residual substance and/or reducing unnecessary loss of the first reaction solution (e.g., filling the conduit of the fluid path system 12) such as a trace amount of the biological sample solution.

In certain embodiments, the method of determining a nucleic acid sequence further comprises performing (prior to the first reaction) the following steps prior to performing step S120: air is introduced into the reaction device 62 through the first flow channel. Thus, before the first reaction is initiated, a section of air is injected to separate the first reaction solution flowing in subsequently from the cleaning solution in the liquid path system, so as to prevent the micro biological sample from being diffused and/or diluted, thereby preventing the nucleic acid molecules to be detected from being connected to the reaction device 62 and the subsequent detection of the nucleic acid molecules to be detected from being influenced.

In certain embodiments, the method of determining a nucleic acid sequence further comprises performing the following steps before performing step S120 and/or before performing step S140 (before performing the first reaction and/or before performing the second reaction): the washing solution in the fourth reservoir 106 is introduced into the reaction device 62 through the first flow path or the second flow path. Thus, the washing solution can clean the reaction device 62, and the subsequent first reaction solution and/or second reaction solution can be prevented from being affected by the last reaction or the residue of the last step.

Referring to FIG. 23, in some embodiments, the reaction device 62 has a solid support surface having a first sequencing primer immobilized thereon, at least one end of the nucleic acid molecule comprises at least a portion of a sequence that is capable of complementary pairing with at least a portion of the first sequencing primer, and the first reaction comprises complementary pairing of at least a portion of the nucleic acid molecule with the first sequencing primer for attachment to the reaction device 62. The so-called "first sequencing primer" is an oligonucleotide (a short nucleic acid sequence of known sequence) of known sequence immobilized on the chip surface, often also called a "probe".

In some embodiments, the second reaction solution comprises a first nucleotide, a first polymerase, and a cleavage reagent, and step S140 comprises: a) passing a first nucleotide and a first polymerase into the reaction device 62 through the second flow path and subjecting the reaction device 62 to conditions suitable for a polymerization reaction to bind the first nucleotide to the nucleic acid molecule by extending the first sequencing primer, the first nucleotide comprising a base, a sugar unit, a cleavable blocking group, and a detectable label; (b) exciting the detectable label and collecting a signal from the detectable label; c) passing a cleavage reagent into the reaction device 62 through the second flow path to remove the cleavable blocking group and the detectable label of the first nucleotide; (d) repeating steps (a) - (c) at least once.

In particular, by "subjecting to conditions suitable for a polymerization reaction", temperature conditions are generally involved in addition to the components/reagents required for the polymerization reaction (e.g., polymerase, reaction substrates, i.e., nucleotides, and/or sequencing primers). For example, the nucleic acid sequencing system 90 may further comprise a temperature control system for controlling the temperature of the reaction device 62/chamber to achieve "conditions suitable for polymerization".

The detectable label being, for example, optically detectableThe label to be detected is, for example, a fluorescent molecule. The cleavable blocking group can prevent/inhibit the other nucleotide (first nucleotide) in the reaction system from binding to the next position of the nucleic acid molecule to be detected, and can be a physical block such as a sugar group with an azido group (-N) at the 3' -position₃) It may be a non-physical block (virtual block) such as a steric conformation in which the blocking group can form a barrier to the progress of the extension reaction in the extension reaction solution system.

In certain embodiments, the second reaction solution further comprises a second nucleotide, and step S140 further comprises performing the following after (a): passing a second nucleotide and a first polymerase into the reaction device 62 through the second flow path, and subjecting the reaction device 62 to conditions suitable for a polymerization reaction to bind the second nucleotide to the nucleic acid molecule by continuing to extend the product after step (a), the second nucleotide comprising a base, a sugar unit, and a cleavable blocking group. The second nucleotide is a reversible terminator without a detectable label, and the reaction efficiency of the second nucleotide is generally higher than that of the first nucleotide for the same polymerase compared with the first nucleotide, and this step is performed to facilitate synchronization of the reactions of multiple nucleic acid molecules in a cluster, i.e., to eliminate or reduce the amount of the nucleic acid molecules with a leading (preceding) or lagging (lagging) reaction in a cluster, and to facilitate the sequencing reaction.

Referring to fig. 24, in some embodiments, the second reaction solution comprises a third nucleotide, a fourth nucleotide, a second polymerase, a third polymerase, a cleavage reagent, and a second sequencing primer, and at least one end of the nucleic acid molecule comprises at least a portion of a sequence that is complementary to at least a portion of the second sequencing primer, and passing the second reaction solution through the second flow channel into the reaction device 62 for performing the second reaction comprises: (i) passing a third nucleotide and a second polymerase into the reaction device 62 through the second flow path and subjecting the reaction device 62 to conditions suitable for a polymerization reaction to bind the third nucleotide to the nucleic acid molecule by extending the first sequencing primer to obtain a nascent strand, the third nucleotide being a nucleotide that carries neither a cleavable blocking group nor a detectable label; (ii) passing a fourth nucleotide, a third polymerase and a second sequencing primer through the second flow channel into the reaction device 62 and subjecting the reaction device 62 to conditions suitable for a polymerization reaction to bind the second sequencing primer to the nascent strand and to bind a fourth nucleotide to the nascent strand by extending the second sequencing primer, the fourth nucleotide comprising a base, a sugar unit, a cleavable blocking group and a detectable label; (iii) exciting the detectable label and collecting a signal from the detectable label; (iv) passing a cleavage reagent into the reaction device 62 through the second flow path to remove the cleavable blocking group and the detectable label of the fourth nucleotide; (v) repeating steps (ii) - (iv) at least once. Thus, a second reaction can be achieved by steps (ii) to (v) to achieve sequencing of the nucleic acid molecule.

The third nucleotide can be, for example, a natural nucleotide; the fourth nucleotide may be identical to the first nucleotide; the first to third polymerases can be the same or different, e.g., different types of DNA polymerases or different mutants of the same type of DNA polymerase, each independently capable of binding a specified nucleotide effective to catalyze the performance of a specified extension/polymerization reaction.

Compared with the sequencing method in the previous embodiment, the method can be used for sequencing the sequence of the other end of the nucleic acid molecule to be detected by synthesizing the complementary strand of the nucleic acid molecule to be detected, so as to realize sequencing of the other end of the nucleic acid molecule to be detected.

Similarly, in certain embodiments, the second reaction solution further comprises a fifth nucleotide, and the method further comprises after (ii), passing the fifth nucleotide and a third polymerase through the second flow path into the reaction device 62, and subjecting the reaction device 62 to conditions suitable for a polymerization reaction to bind the fifth nucleotide to the nascent strand by continuing to extend the product after (ii), the fifth nucleotide comprising a base, a sugar unit, and a cleavable blocking group. The fifth nucleotide, for example, the second nucleotide, is a reversible terminator without a detectable label, and this step is performed to facilitate simultaneous reaction of multiple nucleic acid molecules in a cluster, i.e., to eliminate or reduce a certain amount of nucleic acid molecules with a leading (preceding) or lagging (lagging) reaction in a cluster, to facilitate the sequencing reaction, and to obtain longer reads (reads).

It should be noted that the explanations regarding the technical features of the rotary valve 10 and/or the fluid path system 12, such as the structure, connection relationship, operation control, etc., in the above embodiments are also applicable to the method for determining a nucleic acid sequence in any of the embodiments, and the skilled person can understand how to implement the corresponding sequencing method by using and controlling the rotary valve 10 and/or the fluid path system 12 and/or the sequencing system 90 in the above embodiments through the exemplary descriptions of the structure, connection relationship, function, and operation manner of the rotary valve 10 and the related elements/structural components and the current examples of the sequencing method.

Referring to fig. 22, the present application further provides a nucleic acid sequence determination system 90 (sequencing system 90), wherein the nucleic acid sequence determination system 90 includes the fluid path system 12, the detection assembly 92 and the controller 94 according to any of the above embodiments. Illustratively, fluid path system 12 includes a first reservoir 66, a second reservoir 68, rotary valve 10, and reaction device 62. The detection assembly 92 is configured to detect signals from the reaction device 62 during performance of a given reaction, and the controller 94 is configured to control the fluid path system 12 and the detection assembly 92 to perform a method of determining a nucleic acid sequence in any of the embodiments described above.

For example, the controller 94 is configured to rotate the rotary valve 10 provided with the first flow path and the second flow path to the first valve position to communicate the first reservoir 66 and the reaction device 62, the first reservoir 66 carrying the first reaction liquid, the first reaction liquid containing nucleic acid molecules; and configured to cause a first reaction solution to enter the reaction device 62 through the first flow channel to perform a first reaction with the rotary valve 10 in the first valve position, the first reaction comprising coupling at least a portion of the nucleic acid molecules to the reaction device 62; and a second reservoir 68 configured to communicate with the reaction device 62 by rotating the rotary valve 10 to a second valve position, the second reservoir 68 carrying a second reaction solution comprising components required for performing nucleic acid sequencing; and a second flow path configured to allow a second reaction liquid to enter the reaction device 62 through the second flow path to perform a second reaction with the rotary valve 10 in the second valve position, the second reaction including allowing the nucleic acid molecules in the reaction device 62 after the first reaction is performed to interact with the second reaction liquid to perform a polymerization reaction and detecting a signal from the reaction to perform sequencing of the nucleic acid molecules. The nucleic acid sequencing system 90 is a machine such as a sequencer or a sequencing platform.

As another example, the controller 94 is configured to control the pump assembly 14 and the rotary valve 10 to flow the liquid in the reservoir 64 toward the reaction device 62 through the first flow path, and to control the pump assembly 14 and the rotary valve 10 to flow the liquid in the reservoir 64 toward the reaction device 62 through the second flow path, and to control the detection assembly 92 to detect a signal from the reaction device 62 while the liquid in the reservoir 64 flows through the reaction device 62 or after the liquid in the reservoir 64 flows through the reaction device 62.

As another example, the first reservoir 66 carries a biological sample solution, the biological sample solution includes nucleic acid molecules, the second reservoir 68 carries a reaction solution, the reaction solution includes components required for performing a polymerization reaction, the controller 94 is configured to control the pump assembly 14 and the rotary valve 10 to allow the biological sample solution in the first reservoir 66 to enter the reaction device 62 for a first reaction including at least a portion of the nucleic acid molecules coupled to the reaction device 62, and the controller 94 is configured to control the pump assembly 14 and the rotary valve 10 to allow the reaction solution in the second reservoir 68 to enter the reaction device 62 for a second reaction after the first reaction, and to control the detection assembly 92 to collect a signal generated by the second reaction including interaction of the nucleic acid molecules in the reaction device 62 with the reaction solution for the polymerization reaction.

In some embodiments, the second reaction produces an optical signal, and the detection assembly 92 includes an imaging detector that detects the optical signal.

A computer-readable storage medium of an embodiment of the present application stores a program for execution by a computer, the execution of the program comprising performing the method for determining a nucleic acid sequence in any of the above embodiments. It is to be noted that the explanation of technical features in the method for determining a nucleic acid sequence in the above embodiment is also applicable to a computer-readable storage medium for realizing the embodiment, and a detailed description of the computer-readable storage medium of the embodiment of the present application is not made herein.

A system of embodiments of the present application configured to perform the method of determining a nucleic acid sequence of any of the above embodiments. It should be noted that the method for determining a nucleic acid sequence, the rotary valve 10 and/or the liquid path system 12 in the above embodiments are also applicable to the system for implementing the embodiments, and reference may be made to the method for determining a nucleic acid sequence, the rotary valve 10 and/or the liquid path system 12 for implementing the system in the embodiments of the present application, which are not expanded herein.

A computer program product comprising instructions for carrying out the method of any one of the above embodiments when the program is executed by a computer. It is to be noted that the explanation of the technical features in the method for determining a nucleic acid sequence in the above embodiment is also applicable to a computer program product for implementing the embodiment, and a detailed description of the computer program product of the embodiment of the present application is not made herein. A nucleic acid sequence determination system of embodiments of the present application includes the above computer program product.

In the description herein, reference to the description of the terms "certain embodiments," "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The logic and/or steps represented in the flowcharts or otherwise described herein, such as an ordered listing of executable instructions that can be considered to implement logical functions, can be embodied in any computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, processor-containing system, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions. For the purposes of this description, a "computer-readable storage medium" can be any means that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

In addition, each functional unit in the embodiments of the present invention may be integrated into one processing module, or each unit may exist alone physically, or two or more units are integrated into one module. The integrated module can be realized in a hardware mode, and can also be realized in a software functional module mode. The integrated module, if implemented in the form of a software functional module and sold or used as a stand-alone product, may also be stored in a computer readable storage medium.

Although embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and that variations, modifications, substitutions and alterations of the above embodiments may be made by those of ordinary skill in the art within the scope of the present application, which is defined by the claims and their equivalents.

Claims

1. A method for determining a nucleic acid sequence, comprising:

rotating a rotary valve provided with a first flow channel and a second flow channel to a first valve position so as to communicate a first storage device and a reaction device, wherein the first storage device carries a first reaction liquid, and the first reaction liquid contains nucleic acid molecules;

with the rotary valve in the first valve position, passing the first reaction liquid through the first flow channel into the reaction device to perform a first reaction comprising attaching at least a portion of the nucleic acid molecule to the reaction device;

rotating the rotary valve to a second valve position to communicate a second reservoir and the reaction device, the second reservoir carrying a second reaction solution comprising components required for performing nucleic acid sequencing;

allowing the second reaction solution to enter the reaction device through the second flow channel with the rotary valve in the second valve position to perform a second reaction, the second reaction including allowing a nucleic acid molecule in the reaction device after performing the first reaction to interact with the second reaction solution to perform a polymerization reaction and detecting a signal from the reaction to perform sequencing of the nucleic acid molecule;

optionally, further comprising after performing the first reaction and before performing the second reaction:

allowing a third reaction solution in a third storage to enter the reaction device through the first flow channel or the second flow channel to perform a third reaction, wherein the third reaction comprises allowing a nucleic acid molecule in the reaction device after the first reaction is performed to interact with the third reaction solution so as to realize amplification of the nucleic acid molecule, and the third reaction solution comprises a component required for amplification;

optionally, further comprising, prior to performing the first reaction:

passing the wash solution in the fourth reservoir through the first flow path into the reaction device;

optionally, further comprising, prior to performing the first reaction:

introducing air into the reaction device through the first flow channel;

optionally, further comprising prior to performing the first reaction and/or prior to performing the second reaction:

and (c) allowing the washing solution in the fourth storage to enter the reaction device through the first flow channel or the second flow channel.

2. The method of claim 1, wherein the reaction device has a solid support surface having immobilized thereon a first sequencing primer, at least one end of the nucleic acid molecule comprises at least a portion of a sequence capable of complementary pairing with at least a portion of the first sequencing primer, and the first reaction comprises complementary pairing of at least a portion of the nucleic acid molecule with the first sequencing primer for ligation into the reaction device;

optionally, the second reaction solution comprises a first nucleotide, a first polymerase and a cleavage reagent, and the passing the second reaction solution through the second flow channel into the reaction device to perform a second reaction comprises:

(a) passing the first nucleotide and the first polymerase into the reaction device through the second flow path and subjecting the reaction device to conditions suitable for a polymerization reaction to bind the first nucleotide to the nucleic acid molecule by extending the first sequencing primer, the first nucleotide comprising a base, a sugar unit, a cleavable blocking group, and a detectable label;

(b) exciting the detectable label and collecting a signal from the detectable label;

(c) passing the cleavage reagent into the reaction device through the second flow path to remove the cleavable blocking group and detectable label of the first nucleotide;

(d) repeating (a) - (c) at least once;

optionally, the second reaction solution further comprises a second nucleotide, the second reaction solution is caused to enter the reaction device through the second flow channel to perform a second reaction, further comprising after (a),

passing the second nucleotide and the first polymerase into the reaction device through the second flow path and subjecting the reaction device to conditions suitable for a polymerization reaction to bind the second nucleotide to the nucleic acid molecule by continuing to extend the product after (a), the second nucleotide comprising a base, a sugar unit, and a cleavable blocking group;

optionally, the second reaction solution comprises a third nucleotide, a fourth nucleotide, a second polymerase, a third polymerase, a cleavage reagent, and a second sequencing primer, at least one end of the nucleic acid molecule comprises at least a portion of a sequence that is capable of complementary pairing with at least a portion of the second sequencing primer, and passing the second reaction solution through the second flow channel into the reaction device for a second reaction comprises:

(i) allowing the third nucleotide and the second polymerase to enter the reaction device through the second flow path and subjecting the reaction device to conditions suitable for a polymerization reaction to bind the third nucleotide to the nucleic acid molecule by extending the first sequencing primer to obtain a nascent strand, the third nucleotide being a nucleotide bearing neither a cleavable blocking group nor a detectable label;

(ii) passing the fourth nucleotide, the third polymerase, and the second sequencing primer into the reaction device through the second flow channel and subjecting the reaction device to conditions suitable for a polymerization reaction to bind the second sequencing primer to the nascent strand and to bind the fourth nucleotide to the nascent strand by extending the second sequencing primer, the fourth nucleotide comprising a base, a sugar unit, a cleavable blocking group, and a detectable label;

(iii) exciting the detectable label and collecting a signal from the detectable label;

(iv) passing the cleavage reagent into the reaction device through the second flow path to remove the cleavable blocking group and detectable label of the fourth nucleotide;

(v) (iii) repeating (ii) - (iv) at least once;

optionally, the second reaction solution further comprises a fifth nucleotide, and the second reaction solution is caused to enter the reaction device through the second flow channel to perform a second reaction, further comprising after (ii),

(iii) passing the fifth nucleotide and the third polymerase into the reaction device through the second flow path and subjecting the reaction device to conditions suitable for a polymerization reaction to bind the fifth nucleotide to the nascent strand by continued extension of the product after (ii), the fifth nucleotide comprising a base, a sugar unit and a cleavable blocking group.

3. A nucleic acid sequencing system comprising, in connection, a fluid path system, a detection assembly and a controller, the fluid path system comprising a fluidic network and a pump assembly in communication with the fluidic network, the fluidic network comprising a first reservoir, a second reservoir, a rotary valve and a reaction device, the rotary valve being provided with a first flow channel and a second flow channel, the first flow channel communicating the first reservoir and the reaction device, the second flow channel communicating the second reservoir and the reaction device, the detection assembly being configured to detect a signal from the reaction device during performance of a specified reaction, the controller being configured to control the fluid path system and the detection assembly to perform the steps of the method of any one of claims 1-2.

4. The system of claim 3, wherein the rotary valve is provided with a common port, a plurality of first ports, a plurality of second ports, and a plurality of communication channels selectively communicating the common port with the first ports or the first and second ports;

when the rotary valve is in the first valve position, the first port and the second port are communicated with each other through the communication groove to form the first flow passage;

the first port and the common port communicate with each other through the communication groove to form the second flow passage when the rotary valve is in the second valve position;

optionally, the rotary valve comprises a stator provided with the common port, the plurality of first ports and the plurality of second ports, and a rotor provided with at least a portion of the plurality of communication grooves, disposed opposite the stator;

optionally, the rotary valve comprises a stator and a rotor disposed opposite the stator, the rotor being provided with the common port, the plurality of first ports and the plurality of second ports, the stator being provided with at least a portion of the plurality of communication grooves;

optionally, the communication groove comprises a first communication groove and a second communication groove which can be communicated, and the first communication groove and the second communication groove are respectively arranged on the rotor and the stator;

optionally, one of the communication grooves is composed of one of the first communication grooves and one of the second communication grooves, one of the first communication grooves has two ends, one end of each of the second communication grooves communicates with one of the first ports, and the other end of each of the second communication grooves selectively communicates with one of the second ports, one of the second communication grooves has two ends, one end of each of the second communication grooves communicates with the common port, and the other end of each of the second communication grooves selectively communicates with one of the first ports;

optionally, the stator comprises a stator end face, the rotor comprises a rotor end face, the stator end face and the rotor end face are attached, the first communicating groove is formed in the rotor end face, and the second communicating groove is formed in the stator end face;

optionally, the plurality of first ports and the plurality of second ports are each disposed around the common port;

optionally, the rotary valve has a central shaft, and the plurality of first ports are arranged at intervals on a circular plane taking the central shaft as a shaft center; and/or

The second ports are arranged on a circular plane taking the central shaft as a shaft axis at intervals;

optionally, the plurality of first ports and the plurality of second ports are located on the same circular plane;

optionally, the plurality of first ports are arranged at intervals along the circumferential direction of the stator or the rotor; and/or

The plurality of second ports are arranged at intervals along the circumferential direction of the stator or the rotor;

optionally, the plurality of first ports and the plurality of second ports are located on the same circumference;

optionally, the rotary valve comprises a valve body in which the stator and the rotor are both housed;

optionally, the rotary valve comprises a valve head covering the stator and disposed on the valve body, the valve head having a first port in communication with the common port, a second port in communication with the first port, and a third port in communication with the second port;

optionally, the rotary valve comprises a positioning structure for positioning the valve head and the stator;

optionally, the positioning structure comprises positioning pins inserted in the valve head and the stator;

optionally, the rotary valve further comprises a driving member connected with the rotor, the driving member is arranged on the valve body, and the driving member is used for driving the rotor to rotate;

optionally, the common port, the plurality of first ports, and the plurality of second ports are isolated from one another when the rotary valve is in the third valve position.

5. The system of claim 4, wherein the rotary valve is disposed upstream of the reaction device.

6. A computer-readable storage medium storing a program for execution by a computer, execution of the program comprising performing the method of any one of claims 1-10.

7. A system configured to perform the method of any of claims 1-2.

8. A computer program product comprising instructions for implementing the method of any one of claims 1-2 when said program is executed by a computer.

9. A nucleic acid sequence determination system comprising the computer program product of claim 8.

10. A nucleic acid sequence determination system, comprising:

the first control module is used for rotating a rotary valve provided with a first flow channel and a second flow channel to a first valve position so as to communicate a first storage and the reaction device, wherein the first storage bears a first reaction liquid, and the first reaction liquid contains nucleic acid molecules;

a second control module for causing the first reaction solution to enter the reaction apparatus through the first flow channel to perform a first reaction including coupling at least a portion of the nucleic acid molecule to the reaction apparatus with the rotary valve in the first valve position;

a third control module, configured to rotate the rotary valve to a second valve position to communicate a second reservoir and the reaction apparatus, wherein the second reservoir carries a second reaction solution, and the second reaction solution contains components required for nucleic acid sequencing;

and the fourth control module is used for enabling the second reaction liquid to enter the reaction device through the second flow channel to carry out a second reaction under the condition that the rotary valve is at the second valve position, wherein the second reaction comprises the steps of enabling the nucleic acid molecules in the reaction device after the first reaction is carried out to interact with the second reaction liquid to carry out a polymerization reaction and detecting signals from the reaction so as to realize the sequencing of the nucleic acid molecules.